mu-Opioid receptor stimulation of inositol (1,4,5)trisphosphate formation via a pertussis toxin-sensitive G protein

The cellular mechanisms underlying opioid action remain to be fully determined, although there is now growing indirect evidence that some opioid receptors may be coupled to phospholipase C. Using SH-SY5Y human neuroblastoma cells (expressing both mu- and delta-opioid receptors), we demonstrated that fentanyl, a mu-preferring opioid, caused a dose-dependent (EC50 = 16 nM) monophasic increase in inositol (1,4,5)trisphosphate mass formation that peaked at 15 s and returned to basal within 1-2 min. This response was of similar magnitude (25.4 +/- 0.8 pmol/mg of protein for 0.1 microM fentanyl) to that found in the plateau phase (5 min) following stimulation with 1 mM carbachol (18.3 +/- 1.4 pmol/mg of protein), and was naloxone-, but not naltrindole- (a delta antagonist), reversible. Further studies using [D-Ala2, MePhe4, Gly(ol)5]enkephalin and [D-Pen2,5]enkephalin confirmed that the response was specific for the mu receptor. Incubation with Ni2+ (2.5 mM) or in Ca(2+)-free buffer abolished the response, as did pretreatment (100 ng/ml for 24 h) with pertussis toxin (control plus 0.1 microM fentanyl, 26.9 +/- 1.5 pmol/mg of protein; pertussis-treated plus 0.1 microM fentanyl, 5.1 +/- 1.3 pmol/mg of protein). In summary, we have demonstrated a mu-opioid receptor-mediated activation of phospholipase C, via a pertussis toxin-sensitive G protein, that is Ca(2+)-dependent. This stimulatory effect of opioids on phospholipase C, and the potential inositol (1,4,5)trisphosphate-mediated rises in intracellular Ca2+, could play a part in the cellular mechanisms of opioid action.